This invention concerns methods for the preparation, purification and isolation of antiviral disulfonic acid, disodium salt, particularly 4xe2x80x2,4-bis-{4,6-bis-[3-(bis-carbamoyl-methyl-1-sulfamoyl)-phenylamino]-[1,3,5]triazin-2-ylamino}-biphenyl-2,2xe2x80x2-disulfonic acid, as well as intermediates useful for its synthesis.
Ellestad et al, J. Med. Chem. 41, 2671 (1998) and U.S. Pat. No. 5,852,015 describe a method for preparation and purification of 4xe2x80x2,4-bis-{4,6-bis-[3-(bis-carbamoyl-methyl-1-sulfamoyl)-phenylamino]-[1,3,5]triazin-2-ylamino}-biphenyl-2,2xe2x80x2-disulfonic acid. The intermediates of this disclosed process, when used in subsequent steps lead to an approximately 70% product purity by HPLC, which requires extensive reverse phase chromatographies, followed by lyophilization to remove residual solvents. The process of Ellestad et al. may be summarized in the following Scheme I. 
The use of pure intermediates leads to a higher purity crude 4xe2x80x2,4-bis-{4,6-bis-[3-(bis-carbamoyl-methyl-1-sulfamoyl)-phenylamino]-[1,3,5]triazin-2-ylamino}-biphenyl-2,2xe2x80x2-disulfonic acid. Formation of impurities that require chromatography to remove can be further reduced by performing the last reaction step at a temperature range of 60-75xc2x0 C. A purity of better than 80% can be obtained. It is desirable to have a process by which even greater purity can be achieved.
This invention provides processes by which a higher purity product can now be achieved with better than 97% purity by precipitation/crystallization from a volume of acetonitrile:water, without the use of tedious or costly chromatographies and lyophilizations. Preferably the volume of acetonitrile:water comprises a mixture ratio of from about 0.75:2 to about 1.5:2, more preferably from about 0.8:2 to about 1.2:2, most preferably about 1:2.
This invention provides a process for the production of 2-[Carbamoylmethyl-(3-nitro-benzenesulfonyl)-amino]acetamide, the method comprising reacting 2-(3-Nitro-benzenesulfonylamino)-acetamide with ClCH2CONH2 in the presence of an aprotic solvent, such as N,N-Dimethylformamide (DMF), and a base. Commercially available bases, such as N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-1,8-naphthalenediamine, sodium carbonate, potassium carbonate or sodium bicarbonate, may be utilized. The pH of useful aqueous reaction medium is preferably maintained as either neutral or slightly acidic, preferably a pH range of from about 6 to about 7, more preferably from about 6.5. In non-aqueous media a stoichiometric excess of base may be utilized. Other useful bases include, but are not limited to, sodium hydride, potassium hydride and KOH in mixtures of alcohol(s) and water.
This procedure for the preparation of the intermediate 2-[Carbamoylmethyl-(3-nitro-benzenesulfonyl)-amino]acetamide may be further characterized as comprising an initial step of preparing 2-(3-Nitro-benzenesulfonylamino)-acetamide by reacting 3-Nitro-benzenesulfonyl chloride with aminoglycine hydrochloride or its free base in the presence of a base. The pH of the reaction medium can be created using commercially available bases, such as sodium carbonate or sodium bicarbonate. This step is preferably carried out at a pH of from about 5 to about 8, more preferably from about 6.5 to about 7.
This invention further provides a process for the synthesis of 4xe2x80x2,4-bis-{4,6-bis-[3-(bis-carbamoyl-methyl-1-sulfamoyl)-phenylamino]-[1,3,5]triazin-2-ylamino}-biphenyl-2,2xe2x80x2-disulfonic acid, the process comprising the steps of:
a) reacting 2-(3-Nitro-benzenesulfonylamino)-acetamide with ClCH2CONH2 in the presence of N,N-Dimethylformamide and a base to provide 2-[Carbamoylmethyl-(3-nitro-benzenesulfonyl)-amino]acetamide;
b) treating the 2-[Carbamoylmethyl-(3-nitro-benzenesulfonyl)-amino]acetamide product of step a) with a reducing agent to provide 2-[(3-Amino-benzenesulfonyl)-carbamoylmethyl-amino]acetamide;
c) treating the 2-[(3-Amino-benzenesulfonyl)-carbamoylmethyl-amino-acetamide product of step b) with cyanuric chloride, 2,4,6-trichloro-1,3,5-triazine, to give 2-[(4-{4-[4-(Bis-carbamoylmethyl-sulfamoyl)-benzyl]-6-chloro-[1,3,5]triazin-2-ylmethyl}-benzenesulfonyl)-carbamoylmethyl-amino]acetamide; and
d) reacting the 2-[(4-{4-[4-(Bis-carbamoylmethyl-sulfamoyl)-benzyl]-6-chloro-[1,3,5]triazin-2-ylmethyl}-benzenesulfonyl)-carbamoylmethyl-amino]acetamide product of step c) with the disodium salt of 4,4xe2x80x2-diamino-2,2xe2x80x2-biphenyldisulfonic acid.
The resulting product of this process may be converted to pharmaceutically acceptable salts thereof by methods known in the art.
More specifically, the process described herein differs from the processes described in J. Med. Chem. 41, 2671 (1998) and in U.S. Pat. No. 5,852,015, outlined in Scheme I, above, in the preparation of intermediate 3, N,Nxe2x80x2-bisacetamido-3-nitrobenzenesulfonamide (see examples for detailed procedures). 
As depicted in Scheme II, m-Nitrobenzenesulfonyl chloride can be reacted with commercially available aminoglycine hydrochloride or its free base in aqueous alkali medium to afford intermediate 2, 2-(3-Nitro-benzenesulfonylamino)-acetamide, in high yields. Commercially available and art recognized bases may be used to prepare the alkali medium. Preferred bases are sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, potassium bicarbonate, sodium hydroxide, and triethylamine. Intermediate 2 can be further substituted by condensation with an xcex1-haloacetamide in aqueous alkali medium to give intermediate 3, 2-[carbamoylmethyl-(3-nitro-benzenesulfonyl)-amino]-acetamide. Chloroacetamide is preferred due to large scale availability and cost. Preferred bases are the alkali carbonates, such as potassium carbonate or sodium carbonate.
While the remainder of the steps in the process are the same as in Scheme I, the quality of the subsequent intermediates and crude 4xe2x80x2,4-bis-{4,6-bis-[3-(bis-carbamoyl-methyl-1-sulfamoyl)-phenylamino]-[1,3,5]triazin-2-ylamino}-biphenyl-2,2xe2x80x2-disulfonic acid, disodium salt have been much improved, permitting the use of simple isolation and purification techniques.
Nitro intermediate 3 is subjected to reduction conditions to afford the amino intermediate 4, 2-[(3-Amino-benzenesulfonyl)-carbamoylmethyl-amino]acetamide. This reduction may be accomplished using reducing agents known in the art, including iron/acetic acid, iron/HCl, granular tin/HCl, SnCl2/HCl, or H2S in aqueous or alcoholic ammonia. The preferred method is catalytic reduction, more specifically using palladium on carbon catalyst in DMF. Use of acid in the reduction has been eliminated, thus simplifying isolation. Without a recrystallization the quality of this intermediate has been raised to approximately 99% by the present procedure, further described in Example 3. 
The condensation of intermediate 4 with cyanuric chloride to give intermediate 6, 2-[(4-{4-[4-(Bis-carbamoylmethyl-sulfamoyl)-benzyl]-6-chloro-[1,3,5]triazin-2-ylmethyl}-benzenesulfonyl)-carbamoylmethyl-amino]acetamide, as described in J. Med. Chem. 41, 2671 (1998) and in U.S. Pat. No. 5,852,015, requires a reaction vessel or container temperature of 100-120xc2x0 C. and the pH to be kept at 6.5-7.2 by the use of a phosphate buffer. It has been found that this condensation can be done without consideration of pH and the use of buffers. Also, the reaction may be carried out at a much lower temperatures in 1-methyl-2-pyrrolidinone and in the presence of sodium carbonate. This reaction can be completed for example at a temperature of from about 10xc2x0 C. to about 90xc2x0 C., more preferably at a temperature of from about 10xc2x0 C. to about 40xc2x0 C., most preferably from about 20xc2x0 C. to about 25xc2x0 C. The isolated penultimate intermediate 6 optionally can be further purified by recrystallization from 1-methyl-2-pyrrolidinone-water mixture.
This invention further comprises a process for the purification of intermediate 6, 2-[(4-{4-[4-(Bis-carbamoylmethyl-sulfamoyl)-benzyl]-6-chloro-[1,3,5]triazin-2-ylmethyl}-benzenesulfonyl)-carbamoylmethyl-amino]acetamide, the process comprising dissolving an amount of intermediate 6 in a volume of water and 1-methyl-2-pyrrolidinone, followed by addition of excess water to precipitate a more purified amount of intermediate 6. Preferably, the ratio of water:1-methyl-2-pyrrolidinone into which the amount of intermediate 6 is dissolved is from about 1:1. Precipitation of the desired product can then be completed by adding additional water to create a water:1-methyl-2-pyrrolidinone ratio of up to about 6:1 (wt:wt), more preferably from 3:1 to about 5:1 (wt:wt), more preferably about 4:1 (wt:wt).
The final step requires a condensation of intermediate 6 with the disodium salt of 4,4xe2x80x2-diamino-2,2xe2x80x2-biphenyldisulfonic acid 5 in the presence of a base, such as Hunig""s base (diisopropylethylamine) or another trialkylamine base, such as tributylamine or triethylamine. J. Med. Chem. 41, 2671 (1998) and U.S. Pat. No. 5,852,015 teaches that this reaction requires a temperature of over 100xc2x0 C. to displace the chlorine of intermediate 6. While this reaction may be conducted at temperatures of up to about 120xc2x0 C., it has been found that this reaction can best be done at a lower temperature. By performing this reaction at a lower temperature impurities that hinder purification are minimized and 4xe2x80x2,4-Bis-{4,6-bis-[3-(bis-carbamoyl-methyl-1-sulfamoyl)-phenylamino]-[1,3,5]triazin-2-ylamino}-biphenyl-2,2xe2x80x2-disulfonic acid, disodium salt can be obtained without the use of chromatographies and lyophilizations. This step may be conducted at a temperature of from about 15xc2x0 C. or higher, preferably about 20xc2x0 C. or higher and most preferably at a temperature of from about 50xc2x0 C. or higher, up to about 100xc2x0 C., more preferably up to 90xc2x0 C., and even more preferably up to a temperature of about 80xc2x0 C. The most preferred temperature range is from about 60xc2x0 C. to 75xc2x0 C. The preferred solvent is dimethyl sulfoxide (DMSO). 4xe2x80x2,4-Bis-{4,6-bis-[3-(bis-carbamoyl-methyl-1-sulfamoyl)-phenylamino]-[1,3,5]triazin-2-ylamino}-biphenyl-2,2xe2x80x2-disulfonic acid, disodium salt produced by this process is typically of 80-85% purity by HPLC. It can be further purified by precipitation/-crystallization from acetonitrile-water (1:2 v/v) and filtration at an elevated temperature, such as from about 30xc2x0 C. to about 60xc2x0 C., preferably at a temperature of about 50xc2x0 C. In this process the compound can be dissolved in a volume of acetonitrile/water of about 1:2 (v:v), followed by addition of additional acetonitrile until crystallization of the compound is achieved, preferably at a final concentration of acetonitrile:water of about 2:1.